Suppression of the expression of particular genes is an important tool both for research and for the development of genetically engineered organisms more fitted for a particular purpose. Gene silencing can be accomplished by the introduction of a transgene corresponding to the gene of interest in the antisense orientation relative to its promoter (see, e.g., Sheehy et al., Proc. Nat'l Acad. Sci. USA 85:8805–8808 (1988); Smith et al., Nature 334:724–726 (1988)), or in the sense orientation relative to its promoter (Napoli et al., Plant Cell 2:279–289 (1990); van der Krol et al., Plant Cell 2:291–299 (1990); U.S. Pat. No. 5,034,323; U.S. Pat. No. 5,231,020; and U.S. Pat. No. 5,283,184), both of which lead to reduced expression of the transgene as well as the endogenous gene.
Posttranscriptional gene silencing has been reported to be accompanied by the accumulation of small (20–25 nucleotide) fragments of antisense RNA, which are reported to be synthesized from an RNA template and represent the specificity and mobility determinants of the process (Hamilton & Baulcombe, Science 286:950–952 (1999)). It has become clear that in a range of organisms the introduction of dsRNA (double-stranded RNA) is an important component leading to gene silencing (Fire et al., Nature 391:806–811 (1998); Timmons & Fire, Nature 395:854 (1998); WO99/32619; Kennerdell & Carthew, Cell 95:1017–1026 (1998); Ngo et al., Proc. Nat'l Acad. Sci. USA 95:14687–14692 (1998); Waterhouse et al., Proc. Nat'l Acad. Sci. USA 95:13959–13964 (1998); WO99/53050; Cogoni & Macino, Nature 399:166–169 (1999); Lohmann et al., Dev. Biol. 214:211–214 (1999); Sanchez-Alvarado & Newmark, Proc. Nat'l Acad. Sci. USA 96:5049–5054 (1999)). In plants the suppressed gene does not need to be an endogenous plant gene, since both reporter transgenes and virus genes are subject to posttranscriptional gene silencing by introduced transgenes (English et al., Plant Cell 8:179–188 (1996); Waterhouse et al, supra). However, in all of the above cases, some sequence similarity is required between the introduced transgene and the gene that is suppressed.
In one example, introduction of a sense transgene consisting of the 5′-UTR (“untranslated region”), coding region and 3′-UTR of an ACC oxidase gene under the control of the CaMV 35S promoter resulted in reduced ACC oxidase enzyme activity in 15% of a population of tomato plants (Hamilton et al., Plant J. 15:737–746 (1998); WO98/53083). However, if inverted and sense repeats of part of the 5′-UTR of this ACC oxidase were included in the construct, suppression was observed in 96% of the plants (Hamilton et al., supra). In addition, suppression of another ACC oxidase gene related in sequence to the coding region of the transgene but not to the 5′-UTR of the transgene was suppressed, showing that double-stranded RNA of any part of the transcript targets the entire RNA transcript for degradation. In addition, high frequency and high level posttranscriptional gene silencing have been found by introduction either of constructs containing inverted repeats of the coding regions of virus or reporter genes, or by crossing together plants expressing the sense and antisense transcripts of the coding region of the target gene (Waterhouse et al., Proc. Nat'l Acad. Sci. USA 95:13959–13964 (1998)). Similar results were obtained by expression of sense and antisense transgenes under the control of different promoters in the same plant (Chuang & Meyerowitz, Proc. Nat'l Acad. Sci USA 97:4985–4990 (2000)).
As gene silencing is a powerful tool for regulation of gene expression, both of endogenous genes and of transgenes, improved methods of gene silencing are desired.